Polyploidization, pervasive among higher plant species, enhances adaptation to water deficit, but the physiological and molecular advantages need to be investigated widely. Long non-coding RNAs (lncRNAs) are involved in drought tolerance in various crops. Herein, we demonstrate that tetraploidy potentiates tolerance to drought stress in cassava (Manihot esculenta Crantz). Autotetraploidy reduces transpiration by lesser extent increasing of stomatal density, smaller stomatal aperture size, or greater stomatal closure, and reducing accumulation of H2O2 under drought stress. Transcriptome analysis of autotetraploid samples revealed down-regulation of genes involved in photosynthesis under drought stress, and less down-regulation of subtilisin-like proteases involved in increasing stomatal density. UDP-glucosyltransferases were increased more or reduced less in dehydrated leaves of autotetraploids compared with controls. Strand-specific RNA-seq data (validated by quantitative real time PCR) identified 2372 lncRNAs, and 86 autotetraploid-specific lncRNAs were differentially expressed in stressed leaves. The co-expressed network analysis indicated that LNC_001148 and LNC_000160 in autotetraploid dehydrated leaves regulated six genes encoding subtilisin-like protease above mentioned, thereby result in increasing the stomatal density to a lesser extent in autotetraploid cassava. Trans-regulatory network analysis suggested that autotetraploid-specific differentially expressed lncRNAs were associated with galactose metabolism, pentose phosphate pathway and brassinosteroid biosynthesis, etc. Tetraploidy potentiates tolerance to drought stress in cassava, and LNC_001148 and LNC_000160 mediate drought tolerance by regulating stomatal density in autotetraploid cassava.

Cassava is a crucial crop that makes a significant contribution to ensuring human food security. However, high-quality telomere-to-telomere cassava genomes have not been available up to now, which has restricted the progress of haploid molecular breeding for cassava. In this study, we constructed two nearly complete haploid resolved genomes and an integrated, telomere-to-telomere gap-free reference genome of an excellent cassava variety, ‘Xinxuan 048’, thereby providing a new high-quality genomic resource. Furthermore, the evolutionary history of several species within the Euphorbiaceae family was revealed. Through comparative analysis of haploid genomes, it was found that two haploid genomes had extensive differences in linear structure, transcriptome features, and epigenetic characteristics. Genes located within the highly divergent regions and differentially expressed alleles are enriched in the functions of auxin response and the starch synthesis pathway. The high heterozygosity of cassava ‘Xinxuan 048’ leads to rapid trait segregation in the first selfed generation. This study provides a theoretical basis and genomic resource for molecular breeding of cassava haploids.